Heart failure is the leading cause of death for both men and women in the United States. The underlying molecular and cellular mechanisms of heart failure are very complex and poorly understood. Key Myofilament Regulatory Proteins (KMRPs), which include cardiac troponin (cTn), tropomyosin (Tm), myosin regulatory light chain 2 (RLC2) and cardiac myosin binding protein C (cMyBP-C), play essential roles in cardiac contractility. The hypothesis is that both extrinsic and intrinsic stresses trigger the molecular signaling processes that result in altered modifications to KMRPs leading to contractile dysfunction and eventually heart failure. Recent studies show convincingly that altered modifications in cTnI and cMyBP-C are directly linked to cardiac dysfunction. An unbiased and systematic analysis of the KMRPs to globally detect the changes in protein modifications, identify which sites are modified or altered, and elucidate how these alterations act in concert during the transition to the heart failure is of paramount importance for the understanding of the underlying molecular mechanisms. However, this remains a major challenge. To address this challenge, we propose to establish a simple and robust top-down mass spectrometry (MS)-based disease proteomics platform to examine KMRPs extracted from both normal and diseased tissues to establish a correlation between altered modifications of KMRPs and cardiac dysfunction. Top-down MS directly analyzes intact proteins providing a bird's eye view to observe all possible modifications simultaneously in one spectrum, which is much more reliable than measuring the proteolytically-digested peptides in the conventional bottom-up approach. The integrated top-down proteomics platform will provide a comprehensive tool to effectively separate the intact KMRPs extracted from myocardial tissues, globally detect all modifications that reflect extrinsic and intrinsic stresses, 3) identify and quantify (novel) modifications, and identify multiple concerted alterations in KMRPs and the changes in the distribution of PTMs among multiple targeted sites during the transition to the end- stage heart failure. The specific aims include: 1) Establish an integrated top-down disease proteomics technology for the separation and characterization of intact KMRPs with high efficiency, sensitivity and simplicity. 2) Determine altered protein modifications in KMRPs from hypertrophied and failing swine myocardium. 3) Determine the functional effects of protein kinase A (PKA) and protein kinase A (PKC)- mediated phosphorylation in KMRPs of normal and diseased swine myocardium. 4) Determine the functional consequence of one novel alteration in KMRPs, e.g. cTn, in regulating cardiac contractility. The success of this research project, which integrates proteomics and functional studies, will provide a global map of protein modifications occurring to the KMRPs under normal and diseased conditions and shed new insights into the molecular mechanism of contractile dysfunction in heart failure.